August 2017⎪Vol. 27⎪No. 8

J. Microbiol. Biotechnol. (2017), 27(8), 1401–1408https://doi.org/10.4014/jmb.1705.05003 Research Article jmbReviewAntiamoebic Activity of Petiveria alliacea Leaves and Their MainComponent, Isoarborinol Lizeth M. Zavala-Ocampo1, Eva Aguirre-Hernández2, Nury Pérez-Hernández1, Gildardo Rivera3, Laurence A.

Marchat1, and Esther Ramírez-Moreno1*

1Posgrado en Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Mexico City 07320, Mexico2Laboratorio de Fitoquímica, Facultad de Ciencias, Universidad Nacional Autónoma de Mexico, Mexico City 04510, Mexico 3Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Tamaulipas 88710, Mexico

Introduction

Medicinal plants are considered as an important source

of chemotherapeutic agents and useful structures for the

development of more effective drugs. Among a variety of

plants that have been studied, Petiveria alliacea (Phytolacaceae)

is extremely interesting because of its multiple biological

activities. P. alliacea is a shrub widely distributed throughout

America and some regions of Africa. Notably, it grows in

several parts of Mexico where it is known by many popular

names, such as hierba del zorrillo, hoja de zorrillo, epazote de

zorrillo, zorrillo, payché, hierba de las gallinitas, or sanituwan

[1]. Previous phytochemical characterization of P. alliacea

revealed the presence of triterpenoids, saponins, polyphenols,

coumarins, benzaldehyde, benzoic acid, flavonoids, fredelinol,

pinitol, and allantoin [2-5].

P. alliacea has a broad range of therapeutic properties. It

is used as an antileukemic, antispasmodic, antirheumatic

(topical use), anticancer, immunostimulant, anti-inflammatory,

antinociceptive, and antimicrobial agent. Ethnobotanical

data indicate that P. alliacea leaves are used in Guatemala

for the treatment of dysentery and protozoal infections [6, 7].

In Mexico, it is also used to treat diseases as diverse as the

cold, rabies, and dysentery, as well as stomach ache [1, 8]. The

antiprotozoal properties of P. alliacea have been proved in vitro

by Berger et al. [6] and Cáceres et al. [7] who reported its activity

against Trypanosoma cruzi epimastigotes and trypomastigotes.

Additionally, Echevarría and Torres [9] reported the inhibitory

effect of P. alliacea on the growth of Giardia lamblia in vitro.

However, the activity of P. alliacea against Entamoeba histolytica,

the protozoan parasite that is responsible for human

amoebiasis, has not previously been reported.

Received: May 2, 2017

Revised: June 7, 2017

Accepted: June 13, 2017

First published online

June 16, 2017

*Corresponding author

Phone: +52 57 29 60 00;

Fax: +52 55 5729 6000;

E-mail: estherramirezmoreno@

yahoo.com

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2017 by

The Korean Society for Microbiology

and Biotechnology

Petiveria alliacea L. (Phytolacaceae) is a medicinal plant with a broad range of traditional

therapeutic properties, including the treatment of dysentery and intestinal infections caused

by protozoan parasites. However, its effects against Entamoeba histolytica have not been

reported yet. We investigated the antiamoebic activity present in the leaves of P. alliacea

Antiamoebic activity was evaluated in methanolic and aqueous extracts, as well as in the

hexanic, methanolic, and EtOAc fractions. The P. alliacea methanolic extract showed a better

antiamoebic activity than the aqueous extract with an IC50 = 0.51 mg/ml. Likewise, the hexanic

fraction was the most effective fraction, showing a dose-dependent activity against

E. histolytica, with an IC50 = 0.68 mg/ml. Hexanic subfraction 12-19 showed the highest

antiamoebic activity at 0.8 mg/ml, producing 74.3% growth inhibition without any toxicity in

mammal cells. A major component in subfraction 12-19 was identified as isoarborinol, which

produced 51.4% E. histolytica growth inhibition at 0.05 mg/ml without affecting mammal cells.

The P. alliacea leaf extract has antiamoebic activity that can be attributed to a major metabolite

known as isoarborinol.

Keywords: Petiveria alliacea, isoarborinol, antiamoebic activity, cytotoxicity

1402 Zavala-Ocampo et al.

J. Microbiol. Biotechnol.

Materials and Methods

Collection and Identification of P. alliacea

Petiveria alliacea was collected in Catemaco, Veracruz, Mexico in

April and May 2015. Taxonomic identification was confirmed in

the herbarium of the Universidad Autonoma Metropolitana

Xochimilco, Mexico by M. Sc. Aurora Chimal Hernández, and an

herbarium specimen was deposited in the National Herbarium of

Mexico (MEXU) (voucher number 1414464).

Obtaining Extracts and Fractioning

P. alliacea leaves were dried at room temperature. To obtain the

aqueous extract, 20 g of leaves was macerated with 300 ml of

distillated water and concentrated by lyophilization. For methanolic

extract preparation, 1.8 kg of leaves was macerated with 12 L of

methanol and concentrated with a vacuum rotary evaporator.

Then, fractions of methanol extract were obtained by dissolving

133 g of the extract with 50 ml of MeOH and absorbed in silica gel

(0.015-0.04 mm; Macherey-Nagel) to be subsequently fractionated

with 250 ml of n-hexane, EtOAc, and methanol. The hexanic

fraction (6.8 g) was selected and subfractioned through a silica gel

column using 400 ml mixtures of n-hexane:EtOAc (100:0, 90:10,

80:20, 70:30, 60:40, 50:50, 30:70, 20:80) and EtOAc:methanol

(100:0, 80:20, 60:40, 0:100) of increasing polarity, as the mobile

phase. Subfractions of 100 ml were collected and analyzed for

chromatographic profiling (Fig. 1).

Subfraction Analysis

Subfractions were analyzed by thin-layer chromatography

using silica gel plates, and mixtures of n-hexane/toluene, n-

hexane/EtOAc, and chloroform/EtOAc/formic acid as the mobile

phase. The spots were detected using various techniques and

reagents; those with similar profile were mixed and their yield

was calculated. An abundant white precipitate observed in the

subfraction 12-19, was purified through the recrystallization method,

by dissolving it in a methanol/chloroform mixture. The melting

point of the compound was determined and it was analyzed by

nuclear magnetic resonance of hydrogen and carbon (1H-NMR,13C-NMR) in a spectrometer (Bruker, Advance DPX400 MHz) at

300 MHz. The spectrum was compared with previously reported

data to identify the molecule.

Isoarborinol: colorless crystal with mp 296-298oC. 1H NMR

(300 MHz, CDCl3) δ 5.23 (1H, d, J= 6.2 Hz, H-11), 3.22 (1H, dd,

J=10.2, 3.8 Hz, H-3β), 1.03 (3H, s, H-25), 0.98 (3H, s, H-23), 0.89

(3H, d, J= 6.5 Hz, H-29), 0.83 (3H, d, J= 6.8 Hz, H-30), 0.82 (3H, s,

H-24), 0.81 (3H, s, H-26), 0.77 (3H, s, H-27), and 0.76 (3H, s, H-28).13C NMR (71.4 MHz, CDCl3) δ 39.1(C-1), 27.8(C-2), 78.9(C-3),

39.6(C-4), 52.3(C-5), 21.4(C-6), 26.7(C-7), 42.8(C-8), 148.8(C9),

35.9(C-10), 114.3(C-11), 36.0(C-12), 36.8(C-13), 38.2(C-14), 29.7(C-

15), 36.0(C-16), 42.8(C-17), 52.1(C-18), 20.2(C-19), 28.2(C-20),

59.6(C-21), 30.8(C-22), 28.2(C-23), 15.6(C-24), 22.1(C-25), 17.0(C-

26), 15.3(C-27), 14.0(C-28), 22.1(C-29), and 23.0(C-30).

Cell Cultures

E. histolytica HM1-IMSS trophozoites were axenically grown at

37°C in TYI-S-33 medium, supplemented with 20% bovine serum

[10]. Cells were harvested in the log phase of growth for all

experiments. Human epithelial colorectal adenocarcinoma cells

(Caco-2, HTB-37; ATCC, USA) were grown in advanced minimum

essential medium (MEM; Gibco, USA) supplemented with 5%

fetal bovine serum, 200 mM glutamine (Gibco), 0.0125% penicillin,

and 0.02% streptomycin. Cultures were maintained at 37°C with a

humidified atmosphere of 5% CO2.

Antiamoebic Activity

E. histolytica trophozoites (1.5 × 105) were treated with increasing

amounts of P. alliacea leaf extracts, fractions, subfractions, or the

purified compound. Extracts were dissolved in culture medium,

whereas fractions and subfractions were dissolved in poly-

vinylpyrrolidone (PVP), and the purified compound was first

dissolved in chloroform and polyethylene glycol E-4000 (PEG),

and this was followed by evaporation in a speed vacuum and

dissolution on TY1-S-33 culture medium. Cultures were incubated

at 37°C for 48 h. The trophozoite number and viability were

determined [11]. A positive control treated with 0.04 μg/ml of

metronidazole, a negative control without treatment, and a

Fig. 1. Schematic diagram of P. alliacea fractionation. Gray boxes denote subfractions containing the highest concentrations

of metabolites; numbers below indicate the amount of these

subfractions.

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vehicle control treated with DMSO, PVP, or PEG were included in

each experiment. Experiments were performed twice in triplicates.

Data were expressed as the mean ± standard deviation (SD).

Cytotoxicity Assays

Activity of NADPH-dependent cellular oxidoreductase

enzyme. Caco-2 cells were cultured in a 96-well microplate (2.0 ×

104 cells/well) in the presence of different amounts of hexanic

subfractions (0.1 to 0.8 mg/ml) or the purified compound (0.05 to

0.3 mg/ml), for 48 h. Then, cells were incubated with 1 mM MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) at

37°C for 4 h. The medium was removed and the formazan dye

crystals were solubilized in 100 μl of DMSO for 5 min. Cell

viability was determined from absorbance at 570 nm wavelength

as previously described [11]. Experiments were performed twice

in triplicates and results were expressed as the mean ± SD.

Lactate dehydrogenase release. Caco-2 cells were cultured in

the presence of the hexanic subfractions or the pure compound, as

described above. Supernatants were collected, centrifuged at

500 ×g for 5 min, and transferred to a microtiter plate (50 μl/well)

to determine lactate dehydrogenase (LDH) release using the

CytoTox 96 Non-radioactive Cytotoxicity Assay (Promega) following

manufacturer recommendations. Experiments were performed

twice in triplicates and results were expressed as the mean ± SD.

Acute Oral Toxicity

Acute oral toxicity was evaluated in CD1 male mice, weighing

25–30 g (5 mice per group), according to the guidelines for testing

chemicals number 423, Organization for Economic Cooperation

and Development (OECD), and the Mexican official standard for

the production, use and care of laboratory animals (NOM-062-

ZOO-1999). The protocol was approved by the Institutional Ethics

Committee (January 1st 2016). Animals were fasted for 12 h and

they received 2 g/kg hexanic fraction through an oral cannula.

The control group only received saline solution. Then, they were

observed for 1 min to evaluate possible behavioral effects. Toxic

effects such as loss of movement, tremors, convulsions, ataxia,

respiratory arrest, and death were evaluated at 30 min, every 2 h

for 24 h, and every day for 14 days. Additionally, animals were

weighed daily in order to observe weight loss.

Statistical Analyses

All the data were processed using the Graph Pad Prism 6

software. Statistical analyses were performed using one-way

ANOVA and Dunnett’s and Bonferroni tests. Statistical significance

was set at p≤ 0.05.

Results

The P. alliacea Methanolic Extract Has Antiamoebic Effect

To initiate the evaluation of P. alliacea on E. histolytica

trophozoite cultures, we first compared the effects of

aqueous and methanol extracts. The aqueous extract only

had effect at the highest amount tested (1.8 mg/ml),

producing 49.7% growth inhibition, in reference to the

control group treated with culture medium. By contrast,

the methanol extract showed a dose-dependent antiamoebic

activity; surprisingly, the effect decreased when the extract

concentration was increased. The methanol extract showed

an IC50 = 0.51 mg/ml (Fig. 2A).

P. alliacea Fractions Exhibit Differential Activity against

E. histolytica

To better characterize the methanol extract, we fractioned

it to identify the most active fraction. Results showed that

P. alliacea fractions have different effects on E. histolytica

growth. The hexanic fraction had a dose-dependent effect,

producing 60.7% growth inhibition at the major concentration

of 2.1 mg/ml; this fraction had an IC50 of 0.68 mg/ml. The

EtOAc fraction also had a dose-dependent effect, producing

75% trophozoite growth inhibition at the concentration of

2.1 mg/ml; this fraction had an IC50 of 1.28 mg/ml. In

contrast, the methanolic fraction did not show dose-

dependent activity, since all concentrations produced about

50% growth inhibition (Fig. 2B). Even though the three

fractions had antiamoebic effects, we decided to focus our

study on the hexanic fraction that had the lowest IC50 and

produced a clear dose-dependent response.

The P. alliacea Hexanic Fraction Is Not Toxic to Mice

In order to know the potential systemic toxicity of the

hexanic fraction, we performed an acute systemic toxicity

test in CD1 mice, using a single dose of 2 g/kg by oral

route and assessing the general toxic effect within a short

observation period. After administration, animals showed

no sign or symptom of systemic toxicity, mortality, or

macroscopic damage throughout the 14 days of the daily

observation period (data not shown). Additionally, no

significant difference in weight gain was observed, in

comparison with control group animals (Fig. 2C). Thus, the

lethal dose 50% (LD50) was considered higher than 2 g/kg,

which demonstrated the safety of the hexanic fraction

under these experimental conditions.

P. alliacea Subfractions Exhibit a Greater Antiamoebic

Effect than the Hexanic Fraction

To gain insight into the compounds that are responsible for

its antiamoebic effect, the hexanic fraction was subfractioned

using n-hexane, EtOAc, and methanol mixtures. We obtained

106 subfractions, which were clustered according to their

thin-layer chromatography profiles (data not shown).

Subfractions 12-19, 31-38, 51-60, and 87-96, containing the

1404 Zavala-Ocampo et al.

J. Microbiol. Biotechnol.

highest metabolite concentration, were chosen to evaluate

their activity on E. histolytica cultures at two concentrations

(0.1 and 0.8 mg/ml). In comparison with control groups,

subfraction 12-19 showed the highest effect at 0.8 mg/ml,

with 74.3% growth inhibition. The subfraction 31-38

produced 55.3% growth inhibition at 0.1 mg/ml, but its

activity decreased to 30% at 0.8 mg/ml. The subfractions

51-60 and 87-96 produced less than 50% growth inhibition

at the two concentrations tested (Fig. 3A). Subfraction 12-

19, with the highest antiamoebic activity, was selected to

evaluate its cytoxicity on mammal cells.

P. alliacea Subfraction 12-19 Is Not Cytotoxic toward

Mammal Cells

Cytotoxicity toward Caco-2 cells was evaluated using

increasing concentrations of subfraction 12-19, from 0.1 to

0.8 mg/ml. None of the concentrations tested occasioned

significant changes in cell viability compared with the

group without treatment (Fig. 3B). Likewise, this subfraction

did not produce any cellular cytotoxicity in reference with

the group treated with Triton X-100, which caused 100%

cellular lysis (Fig. 3C).

Identification of the Major Compound of Subfraction 12-19

To identify the main component of subfraction 12-19,

we first submitted it to a thin-layer chromatography

analysis. The chromatographic profile was visualized with

p-anisaldehyde, suggesting that the major component

could be a terpenoid (data not shown). The compound was

purified by the recrystallization method, obtaining a total

Fig. 2. Antiamoebic activity and acute toxicity of P. alliacea leaves. E. histolytica growth inhibition by aqueous and methanolic extract (A), and hexanic, EtOAc, and methanolic fractions (B). PVP

(polyvinylpyrrolidone), standard medium (-), or metronidazole (MTZ) at 0.04 µg/ml were included as controls. Data represent the mean of two

independent experiments performed in triplicates ± SD, *p ≤ 0.05 in reference to the negative control. (C) Acute toxicity of the hexanic fraction in

CD1 mice. Mice were treated with a single dose of 2 g/kg hexanic fraction on day 1 and the weight was recorded daily (solid line). Saline solution

was included as the control (dashed line). Data represent the average weight of five mice ± SD. Statistical significance was set at p ≤ 0.05.

P. alliacea and Isoarborinol Antiamoebic Activity 1405

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amount of 68.3 mg. The compound melting point was

calculated as 296-298oC. The structure of the compound

was elucidated by 1H NMR and 13C NMR, and it was

identified as the pentacyclic triterpenoid isoarborinol by

comparison of spectroscopic data with previously reported

spectra [12, 13]. Briefly, the 1H NMR spectrum of isoarborinol

revealed the presence of six singlets at low frequency: 0.76,

0.77, 0.81, 0.82, 0.98, and 1.03 δH assigned to methyl groups

of C-28, C-27, C-26, C-24, C-23, and C-25 δC, respectively.

Furthermore, two doublets at 0.83 and 0.89 δH corresponding

to two methyl groups of the isopropyl fragment and

olefinic and methine signals at 5.23 δH and 3.22 δH for H-11

and H-3 were easily recognized. Additionally, the concordance

of all the signals of 13C NMR with those described previously

Fig. 3. Antiamoebic activity and cytotoxicity of P. alliacea subfractions. (A) Antiamoebic activity of 12-19, 31-38, 51-60, and 87-96 subfractions. PVP (polyvinylpyrrolidone) and metronidazole (MTZ) at 0.04 µg/ml were

included as controls. Data represent the average of two independent experiments performed in triplicates ± SD. **p ≤ 0.05. (B and C) Subfracion

12-19 cytotoxicity was determined in Caco-2 cells. (B) Viability was determined by MTT assay and (C) cytotoxicity was evaluated using the Cyto

Tox 96 Non-radioactive Cytotoxicity Kit. MEM (control −), Triton X-100 (control +), and DMSO were included as controls. Experiments were

performed twice in triplicates. Data corresponding to the mean value ± SD were expressed in percentage in relation to the number of cells grown

in culture medium.

1406 Zavala-Ocampo et al.

J. Microbiol. Biotechnol.

confirmed the isoarborinol structure (Fig. 4A). Copies of

the original spectra are obtainable from the corresponding

author.

Isoarborinol Has Antiamoebic Activity

To confirm that the activity of subfraction 12-19 was due

to isoarborinol, E. histolytica trophozoites were cultured in

the presence of isoarborinol. The cell count showed that

isoarborinol has a dose-dependent antiamoebic activity,

producing 51.4% E. histolytica growth inhibition at 0.05 mg/ml

and 85.2% at 0.3 mg/ml, in reference to control groups

(Fig. 4B). Interestingly, isoarborinol did not produce any

toxicity toward Caco-2 cells since none of the concentrations

used affected cell viability through alteration of mitochondrial

enzyme activity (Fig. 4C) nor caused cell lysis (Fig. 4D).

Discussion

The effect of P. alliacea extracts against the protozoan

parasites T. cruzi and G. lamblia have been previously

reported [6, 7, 9], but this is the first study that reveals its

activity against E. histolytica, the parasite that causes human

Fig. 4. Isoarborinol structure and its effect on E. histolytica and Caco-2 cells. (A) Isoarborinol structure. (B) E. histolytica growth inhibition by isoarborinol. Trophozoites growing in standard medium, polyethylene glycol

(PEG), and metronidazole (MTZ) at 0.04 µg/ml were included as controls. (C and D) Caco-2 cell viability and cytotoxicity in response to

isoarborinol. (C) Cell viability was determined by MTT assay and (D) cytotoxicity was evaluated using the CytoTox 96 Non-Radioctive

Cytotoxicity Kit. MEM (control -) and Triton X-100 (Control +) were included as controls. Experiments were performed twice in triplicates. Data

corresponding to the mean value ± SD are expressed in percentage. **p ≤ 0.05 in reference to the negative control.

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amoebiasis.

Although we chose the P. alliacea hexanic fraction for

further analysis, it is important to mention that the methanol

and EtAOc fractions also have antiamoebic activity, which

suggests that P. alliacea contains metabolites with different

polarity that are active against E. histolytica. These may

include isoarborinol and other triterpenes previously

identified in this plant, such as isoarborinol-acetate and

isoarborinol-cinnamate.

The hexanic fraction of P. alliacea leaves had a dose-

dependent antiamoebic activity, producing 50% growth

inhibition at 0.68 mg/ml. Echevarría and Torres [9]

previously reported this dose-response activity of P. alliacea

methanolic extracts on G. lamblia, finding an IC50 of

2.05 mg/ml. In contrast, P. alliacea seems to be more

effective against trypanosomatids, since Berger et al. [6],

using hexanic and ethanol extracts of P. alliacea found a

marked inhibition of T. cruzi trypomastigotes at IC90 of 285.6

and 692.2 μg/ml, respectively. Additionally Cáceres et al.

[7], reported that the dichloromethane extract of P. alliacea

was active in vitro against epimastigote and trypomastigote

stages of T. cruzi with IC90 = 1.0 mg/ml, which was not

toxic for Artemia salina. In addition, García et al. [14] found

that the ethanolic extract from P. alliacea leaves was active

against the amastigote stage of Leishmania amazonensis with

an IC50 of 151.5 μg/ml. Similarly, the P. alliacea efficiency

has been proved against Plasmodium falciparum. Ruiz et al.

[15] reported an antiplasmodial activity with an IC50>10 μg/ml and they demonstrated that the P. alliacea leaf

hydroalcoholic extract at the IC50 of 3.8 μg/ml was able

to inhibit ferriprotoporphyrin (FP) biomineralization, a

Plasmodium-specific process of heme detoxification in which

FP derived from the digestion of ingested hemoglobin is

converted to hemozoin (b-hematin).

The similarity between the IC50 of P. alliacea extracts

found in E. histolytica and G. lamblia could be related to

similar action mechanisms in these two amitochondriate

protozoa. In the case of trypanosomatides and P. falciparum,

the lower inhibitory concentrations suggest that the

mechanism of action could involve mitochondria.

As we previously mentioned, P. alliacea contains many

secondary metabolites that can produce different biological

effects in humans and microorganisms. In the leaves of

P. alliacea, the presence of steroids, terpenoids (isoarborinol,

isoarborinol-acetate, isoarborinol-cinnamate), saponins,

polyphenols, and tannins has been reported [16], as well as

allantoin, linoleic acid, lignoceryl alcohol, lignoceryc acid,

lignoceryl ester, a-freidelinol, nonadecanoic acid, oleic,

palmitic and stearic acids [4], glucosides, and alkaloids

[17]. However, none of the previous studies identified the

metabolites responsible for the antiprotozoal activity.

Therefore, we decided to investigate which phytochemical

molecules are responsible for the antiamoebic activity of

P. alliacea.

The antiamoebic activity was increased after extract

fractioning, as the early fractions (fraction 12-19) showed

74.3% growth inhibition with 0.8 mg/ml, suggesting that

active metabolites were concentrated in this fraction. Indeed,

we found that it contains isoarborinol (C30H50O), a pentacyclic

triterpenoid whose molecular weight is 426.7 g/mol. It is a

class of 30-carbon isopentenoid (isoprenoid) that can function

like sterols, as structural components of membranes [18].

The antiprotozoal activity of other pentacyclic triterpenoids

has been previously reported. Maslinic acid, a pentacyclic

derivative present in the olive fruit (Olea europaea), is

capable of blocking the entry of Toxoplasma gondii tachyzoites

into the cell and can inhibit some of its proteases. It also

produces gliding motility and ultrastructure alterations in

parasites [19]. Additionally, the antiprotozoal activity of

oleanolic acid and its isomer, ursolic acid, has been

documented in P. falciparum [20-24], Leishmania donovani,

L. major, L. amazonensis [25], L. infantum [26], and T. cruzi

[27, 28].

Isoarborinol has been reported in P. alliacea extracts,

however, its specific antiamoebic activity was not known.

Here, we found that isoarborinol has a high effect against

E. histolytica trophozoites since a concentration of 0.05 mg/ml

produced 51.4% growth inhibition. Although this concentration

is not comparable to the one of metronidazole (IC50 =

0.04 μg/ml), isoarborinol is not toxic toward human cells at

the concentrations effective against amoeba, which could

be an advantage over metronidazole toxicity. Therefore, it

would be interesting to investigate whether the effects of

isoarborinol in amoeba are similar to those produced by

other triterpenes in other protozoan parasites. Forthcoming

investigations will allow us to obtain information regarding

the mechanisms of action of this molecule in E. histolytica

trophozoites.

Acknowledgments

This work was supported by Consejo Nacional de Ciencia

y Tecnología (CONACyT), Secretaría de Investigación y

Posgrado (SIP)-Instituto Politécnico Nacional (IPN), and

Comisión de Operación y Fomento de Actividades Académicas

(COFAA)-IPN, Mexico. We greatly thank Verónica Muñoz

Ocotero and Jacqueline Soto Sánchez for their technical

assistance.

1408 Zavala-Ocampo et al.

J. Microbiol. Biotechnol.

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